Fig 2: Association between soluble JAM-C and full-length JAM-B on ADSCs. Whole-cell lysates (1% of the input protein samples) and the samples immunoprecipitated (IP) with IgG or JAM-B Ab were immunoblotted (IB) with the indicated Abs. N.S., nonspecific signals.

Fig 3: DIDO3?E16 ESC expression alterations detected by RNA-seq, with effects on protein expression.a The Venn diagram depicts the relationship of genes identified for altered expression in DIDO3?E16 ESC compared to WT, and restored in DIDO3?CT+HADIDO3 in RNA-seq (DEXSeq, Supplementary Table 1; 3'UTR + 2 kb, Supplementary Table 2), and a DIDO3 DNA-binding site determined by ChIPseeker of previous ChIP-seq data (Supplementary Table 3); some relevant examples are highlighted. Note: (*) Significant alteration (1.5-fold difference; FDR < 0.05) was not detected by RNA-seq data analysis, but RT-PCR confirmed a prolonged 3'UTR of the transcript only in the DIDO3?E16 mutant ESC. For details, see Results. b GO term enrichment analysis and heatmap of the selected genes. UP- and DOWN regulated genes in DIDO3?E16 versus WT are shown in red and blue, respectively. Significantly enriched GO terms (biological processes) were identified using DAVID (https://david.ncifcrf.gov/), g: Profiler (https://biit.cs.ut.ee/gprofiler/gost), and GSEA (https://www.gsea-msigdb.org/). For additional details of the enrichment analysis, see Supplementary Fig. 1d. c JAM2 example gene; IGV image shows the JAM2 locus, aligned and normalized RNA-seq reads of triplicates of WT, DIDO3?E16, and DIDO3?CT+HADIDO3 ESC as well as DIDO3 ChIP-seq peaks (red) with overlapping RNA pol II S2p peaks (green) and defined 3'UTR regions (blue). d Different abundance of JAM2 short isoform1 and long isoform2 (indicated in c), confirmed in WT, DIDO3?E16, and DIDO3?+HADIDO3 RNA as tested by RT-PCR. e The histogram shows abundance of the JAM2 protein in WT and DIDO3?CT ESC as measured by flow cytometry analysis after anti-JAM2 antibody staining. f CARM1 example gene; parameters as in (b) for the CARM1 locus, focusing on 3'UTR read-throughs in the DIDO3?E16 mutant (red box). g Western blot analysis of lysates of ESC and embryonic bodies without LIF at d2 and d4, comparing CARM1 protein levels in WT, DIDO3?E16, and DIDO3?CT+HADIDO3 cells; ß-ACTIN used as loading control.

Fig 4: Schematic model for the regulation of ADSC functions by soluble JAM-C. The soluble JAM-C (sJAM-C) deposited on the extracellular matrix interacts with JAM-B and partly with JAM-C on ADSC. sJAM-C/JAM-B signaling reaches the nucleus (dashed arrow) and stimulates cellular proliferation and expression of MSC markers (arrows). MSC, mesenchymal stromal/stem cell.

Fig 5: A Soluble JAM-C couples with JAM-B to promote the ADSC adhesion and niche function. (A) Knockdown of the Jam2 gene encoding mouse JAM-B in ADSCs using the CRISPR method. (B) Western blot for the indicated proteins in the whole-cell lysates of the revealed ADSCs. (C) Cell adhesion assay for ADSCs grown in the indicated culture conditions. The relative levels are shown in histograms (mean ± SD; n = 8). (D) RT-qPCR for the indicated MSC markers in ADSCs cultivated in the indicated culture conditions. The relative expression levels are shown in the histograms (mean ± SD; n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001.